InteractiveFly: GeneBrief

squeeze : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - squeeze

Synonyms -

Cytological map position- 91F4-91F4

Function - transcription factor

Keywords - regulates neuropeptidergic cellular identity, regulates FMRFamide

Symbol - sqz

FlyBase ID: FBgn0010768

Genetic map position - 3R: 14,989,943..14,996,296 [+]

Classification - zinc finger

Cellular location - nuclear



NCBI link: EntrezGene

sqz orthologs: Biolitmine
BIOLOGICAL OVERVIEW

One of the most widely studied phenomena in the establishment of neuronal identity is the determination of neurosecretory phenotype, in which cell-type-specific combinatorial codes direct distinct neurotransmitter or neuropeptide selection. However, neuronal types from divergent lineages may adopt the same neurosecretory phenotype, and it is unclear whether different classes of neurons use different or similar components to regulate shared features of neuronal identity. This question was addressed by analyzing how differentiation of the Drosophila larval leucokinergic system, which is comprised of only four types of neurons, is regulated by factors known to affect expression of the FMRFamide neuropeptide. All leucokinergic cells express the transcription factor Squeeze (Sqz). However, based on the effect on Leucokinin (LK) expression of loss- and gain-of-function mutations, three types of LK regulation are described. In the brain LHLK (lateral horn leukokinin) cells, both Sqz and Apterous (Ap) are required for LK expression, but surprisingly, high levels of either Sqz or Ap alone are sufficient to restore LK expression in these neurons. In the s SELK cells, Sqz, but not Ap, is required for LK expression. In the abdominal ABLK neurons, inhibition of retrograde axonal transport reduces LK expression, and although sqz is dispensable for LK expression in these cells, it can induce ectopic leucokinergic ABLK-like cells when over-expressed. Thus, Sqz appears to be a regulatory factor for neuropeptidergic identity common to all leucokinergic cells, whose function in different cell types is regulated by cell-specific factors (Herrero, 2007).

It has been shown that Ap is required for LK expression only in LHLK cells. Ap is also necessary for proper transcription of the Fmrf gene in the thoracic Tv neurons. In attempts to understand the mechanisms underlying leucokinergic differentiation, it was asked whether other factors known to control expression of the FMRFamide neuropeptide, i.e., Sqz and the BMP signalling pathway, affected LK expression. Indeed, the number of LK-immunopositive cells is strongly reduced in sqzlacZ mutant larvae. It has been proposed that Apterous is not necessary for the emergence and maintenance of LHLK cells. Earlier reports have established that the Tv cells are present in sqz mutants, although they do not express Fmrf. Are the LK cells present in sqz mutants? This question could not be directly addressed due to the lack of independent markers for following the fate of the LK cells, but the results presented in this study indicate that in sqz mutants, leucokinergic cells are born, but fail to express LK: (1) LK expression is restored by postmitotic expression of Sqz; (2) LK-immunoreactive cells are detected in sqz mutant brains during early larval stages, and later disappear; (3) the few LK-expressing cells detected in sqz mutants often show very faint immunostaining, which suggests that reduction of Sqz protein decreases Lk transcription but does not affect cell survival. The reduction in the number of leucokinergic cells in sqz mutants even in first instar larvae indicates that sqz is necessary for induction of LK expression, and the weaker phenotype observed in early larval stages may reflect perdurance of the wild type product supplied by the mother, or a requirement for sqz also for maintenance of LK expression (Herrero, 2007).

Although all leucokinergic neurons express sqz, they differ in how this transcription factor affects LK expression. In this study, at least three neuronal types have been identified based on the components that regulate LK expression: (1) in the LHLK neurons, LK expression is controlled by the transcription factors Ap and Sqz; (2) in the SELK neurons, Sqz, but not Ap, is necessary for wild type Lk transcription; (3) in the ABLK neurons, Sqz is dispensable for LK expression even though it can induce leucokinergic ABLK-like cells, and LK expression is affected by inhibition of retrograde axonal transport only in the ABLK cells. Regarding the ALK neurons, the number of this cell type is highly variable, and thus was not further analyzed in this study (Herrero, 2007).

By analyzing two different sqz alleles, it was shown that, besides differences in the components that control LK expression, the amount of Sqz protein required to achieve wild type LK expression also varies. RealTime PCR analysis indicates that the sqzlacZ allele has undetectable levels of sqz transcripts, while the sqzGAL4 allele has reduced, but measurable sqz transcription. Consistent with these results, sqzlacZ mutant larvae show a large reduction in the number of LHLK and SELK cells detected by anti-LK immunostaining, while sqzGAL4 mutants do not affect LK expression in the LHLK neuron, but do show a significant decrease in the number of LK-immunopositive SELK cells. Moreover, restoring neuronal Sqz protein with the elavGAL4 driver can completely rescue LK expression in LHLK neurons in sqzlacZ mutants, but only partially rescue LK in SELK neurons. The differential effect of these two sqz alleles suggests that there is a threshold of Sqz protein below which Lk transcription is prevented, and this threshold is higher in SELK cells than in LHLK cells (Herrero, 2007).

This study has demonstrated that the reduced LK expression observed in the LHLK cells of sqz mutants is fully rescued when over-expressing the lost protein, indicating that this protein is indeed responsible for the observed phenotype. In addition, the data show that LK expression in these cells does not depend on the Gbb/Wit or Babo/Activin signalling cascades, or any extrinsic retrograde signal. Thus, neuropeptidergic identity of the LHLK neurons is controlled by Ap and Sqz, but not BMP. These cells also express the bHLH transcription factor Dimm, which has been shown to act post-tanscriptionally on the regulation of LK expression in these cells. This combination of factors (i.e., Ap, Sqz, Dimm, but not BMP signalling) is different from any of the known codes that regulate the neuropeptidergic differentiation of the peptidergic neurons in the thoracic ap cluster, which includes the Furin 1-expressing Ap-let cells that do not express Sqz, and the Fmrf-expressing Tv cell, which requires extrinsic signalling through Gbb/Wit (Herrero, 2007).

These results demonstrate that high levels of either Ap or Sqz protein alone are sufficient to induce LK expression in the LHLK cells. Thus, based on the cross-rescue experiments, it can be inferred that Sqz can form transcriptionally active complexes with proteins other than Ap to promote Lk expression. Similarly, Ap is able to promote Lk expression when it complexes with proteins other than Sqz. However, wild type levels of LK expression are only achieved when both proteins are present. It has been shown that Sqz and Ap can physically interact, and that both proteins can form separate complexes with Chip. Chip is involved in Lk regulation, because LK expression in LHLK cells is affected when dLMO, a cofactor that binds Chip and prevents Ap action, is expressed in ap-expressing cells. Thus, Ap and Sqz could weakly activate Lk transcription independently by interacting with Chip and/or other transcription factors, but strong activation would require a synergistic interaction mediated by complexes containing both proteins. A similar mechanism is exerted by Brn2 and Otp to stimulate transcription of the corticotrophin-releasing hormone gene in the neuroendocrine hypothalamic neurons, and by Cdx-2 and Brn-4 to activate expression of the proglucagon gene in pancreatic B-cell lines (Herrero, 2007).

It has been reported that Lk transcription in dimm mutants is downregulated in ABLK cells, while LHLK and SELK cells show slightly upregulated Lk transcription but reduced levels of LK peptide. It was found that the ABLK neurons also differ from LHLK and SELK neurons in that Sqz appears to be dispensable for LK expression in these cells. However, neuronal over-expression of Sqz can frequently induce ectopic LK expression in ABLK-like cells in an ap-independent fashion. Likewise, misexpression of the mammalian homeodomain transcription factor Phox2a in the sympathetic ganglion induces ectopic noradrenergic neurons, even though in the absence of Phox2a, sympathetic development is largely normal (Herrero, 2007).

Another peculiarity of the ABLK cells is that LK expression in these cells seems to depend on a retrograde signal, different from BMP, because the number of ABLK LK-expressing cells is reduced when axonal transport is inhibited. Although the degree of reduction is variable, it is indeed highly significant, because as few as two cells can be found when GluedDN is pan-neurally expressed. This variability could be due to partial inhibition of retrograde transport, as suggested by the late pupal lethality of GluedDN-expressing flies, instead of the earlier embryonic lethality of Glued amorphic mutations. In the thoracic ap-cluster, projection of the Tv cell outside the CNS is essential for its response to Gbb and subsequent Fmrf expression. Likewise, ABLK cells are known to project outside the CNS, and this may be essential for them to react to an extrinsic signal important for induction and/or maintenance of LK expression. Alternatively, extrinsic signalling may control guidance and/or targeting of ABLK axons, as suggested by the ectopic leucokinergic varicosities found in the abdominal neuropile of GluedDN-expressing ganglia, and proper axonal targeting may be an important factor in the regulation of LK expression. Further experiments will be necessary to test these hypotheses, and rule out a possible deleterious effect of GluedDN on ABLK viability (Herrero, 2007).

Ectopic leucokinergic cells can be induced by over-expressing Sqz, but not by Ap. Moreover, ectopic LK neurons can also be classified into three different groups: (1) the ectopic LHLK-like cells, which appear to depend on the relative amount of Sqz and Ap; (2) the ABLK-like cells, which require high levels of Sqz expression, but not ap expression, and (3) the ectopic brain cells, which are the only leucokinergic ectopic cells generated by ectopic expression of Sqz. These latter cells must have unidentified components present in other leucokinergic cells that enable them to activate Lk transcription when Sqz is present (Herrero, 2007).

An ectopic LHLK-like cell is present in a small percentage of the sqzGAL4 mutants. Appearance of this ectopic cell requires Ap, because ap is expressed in this cell and its appearance is prevented in the apUGO35 null allele. In the thoracic ganglion, sqz determines the number and identities of cells of the ap cluster, so that in szq null mutants an extra Dimm and Furin-expressing cell was generated in every ap cluster. Attempts to analyze changes in the number of ap and dimm expressing cells in sqz mutants was precluded by the large number of ap cells surrounding the LHLK cell, and the inconsistent expression of the dimm-GAL4 driver c929 in the LHLK cells, in both wild type and mutant brains. However, the results obtained when Sqz and/or Ap were over-expressed in a wild type background suggest a more complex scenario. First, a phenotype was obtained equivalent to that observed in sqzGAL4 mutants, i.e., the appearance of ectopic LHLK-like cells, when Sqz was over-expressed with sqzGAL4 and with the postmitotic ap- and elav- GAL4 drivers. Moreover, these ectopic LK cells disappeared if Ap was coexpressed with Sqz, even though over-expression of Ap alone had no effect whatsoever on LK expression. Based on these data, and on the different levels of Ap protein in cells surrounding the LHLK neuron, it is hypothesized that the relative dose of Ap and Sqz, rather than the absolute amount of Sqz, is essential for specifying the correct number of LHLK cells with detectable LK, and that this process is controlled postmitotically. According to this model, when Sqz is reduced, those cells with low Ap levels would reach an optimum stoichiometric Sqz/Ap ratio leading to ectopic LK expression, while when Sqz is increased, the correct ratio will be present in cells with high Ap levels. In this last case, further increasing Ap would drive the Sqz/Ap ratio away from the optimum for LK expression, and ectopic cells would not be produced (Herrero, 2007).

Two other situations lead to ectopic leucokinergic LHLK-like cells. (1) A small percentage of ectopic LHLK cells was detected in dac mutants. Because Ap has been shown to repress dac expression in the thoracic Tvb cell, it is possible that repression of dac is required to restrict LK expression to the LHLK neuron. (2) Over-expression of the transcription factor Dimm produces ectopic LHLK-like cells, albeit at much higher frequency. Ap also regulates dimm transcription in most cells. Thus, Dimm over-expression might overcome the requirement for a Sqz/Ap optimum ratio to induce LK expression, provided additional necessary factors are also present in the cell. More experiments will be needed to understand the role of Dac and Dimm, and their interaction with Ap and Sqz, in regulating LK expression in the LHLK cell (Herrero, 2007).

Deciphering how cells of different origin acquire the same neurosecretory identity is one of the major challenges in neuronal development. Much of the progress in this field has been achieved by studying the vertebrate catecholaminergic system, in which central and peripheral noradrenergic neurons use a similar combination of factors to specify their neurotransmitter phenotype, but they differ in the hierarchical relations between these factors, and recruit components specific to each neuronal type. It is tempting to speculate that analogous mechanisms may control Lk expression in leucokinergic cells in Drosophila. Consistent with this hypothesis, Sqz, and probably Dimm, appear to be regulatory factors for neuropeptidergic identity common to all leucokinergic cells. However, sqz affects LK expression in each cell subclass in very different ways, and cell-type-specific regulatory modes on Lk have also been described for Dimm. Moreover, additional cell-type-specific factors regulate LK expression in different leucokinergic cells, such as Ap in the LHLK cells, and an unidentified retrograde signal in the ABLK cells. Thus, the leucokinergic neurons comprise a simple, genetically amenable system for understanding how complex regulatory networks confer a similar neurosecretory identity on cells of different origins (Herrero, 2007).


REGULATION
Promoter Structure

The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, it was demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. This study reports the results of in vivo functional analysis of 27 remaining clusters. Transgenic flies were generated carrying each cluster attached to a basal promoter and reporter gene, and assayed embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. The Drosophila pseudoobscura genome was used to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. Conservation of binding-site clustering was incorporated into a new genome-wide enhancer screen, and several hundred new regulatory sequences were predicted, including 85 adjacent to genes with embryonic patterns. It is cocluded that measuring conservation of sequence features closely linked to function -- such as binding-site clusterin -- makes better use of comparative sequence data than commonly used methods that examine only sequence identity (Berman, 2004).

Targets of Activity

Apterous regulates neuropeptide cell identity by the integration of retrograde BMP signaling and a combinatorial transcription factor code

Individual neurons express only one or a few of the many identified neurotransmitters and neuropeptides, but the molecular mechanisms controlling their selection are poorly understood. In the Drosophila ventral nerve cord (VNC), the six Tv neurons express the neuropeptide gene FMRFamide (FMRFa). Each Tv neuron resides within a neuronal cell group specified by the LIM-homeodomain (LIM-HD) gene apterous (ap). The zinc-finger gene squeeze acts in Tv cells to promote their unique axon pathfinding to a peripheral target. There, the BMP ligand Glass bottom boat activates the Wishful thinking receptor, initiating a retrograde BMP signal in the Tv neuron. This signal acts together with apterous and squeeze to activate FMRFamide expression. Reconstituting this 'code,' by combined BMP activation and apterous/squeeze misexpression, triggers ectopic FMRFamide expression in peptidergic neurons. Thus, an intrinsic transcription factor code integrates with an extrinsic retrograde signal to select a specific neuropeptide identity within peptidergic cells (Allan, 2003).

FMRFa is specifically expressed in the six Tv neuroendocrine neurons located bilaterally in the three thoracic (T1-3) segments of the embryonic and larval VNC. apterous is expressed in three interneurons per VNC hemisegment, as well as in a lateral cluster of four neurons (the ap-cluster) in each of the T1-3 hemisegments. One of the four ap-cluster cells is the FMRFa-expressing Tv neuron. All ap interneurons in the VNC, except for the Tv, join a common ipsilateral axon tract termed the ap-fascicle. The Tv axon instead projects to the midline and exits the VNC dorsally to innervate the dorsal neurohemal organ (DNH). The DNH is a club-like neuroendocrine structure formed by two glial cells protruding from the midline of each thoracic segment. Anteriorly, two additional FMRFa-expressing cells are found, denoted SE2 cells. The SE2 cells do not express, nor depend upon, any regulators described in this study for their FMRFa expression. ap is important for the expression of FMRFa in the Tv neurons, but since most ap neurons do not express FMRFa, other regulators are likely needed for FMRFa regulation (Allan, 2003).

To determine whether sqz regulates FMRFa expression, immunoreactivity for the FMRFa peptide was compared in wild-type and sqz mutant L1 larvae. In wild-type, FMRFa immunoreactivity is robust (98%) in all six Tv neurons. In sqz mutants (sqzlacZ/sqzDf), FMRFa staining was found to be reduced in all Tv neurons and was detected in 75% of cells. The T1 segment was most affected, with FMRFa expressed in 40% of T1 Tv neurons. To verify that the observed effects reflected regulation of the FMRFa gene, antibodies recognizing the C-terminal of the FMRFa precursor peptide (proFMRF) were used, as well as an FMRFa-lacZ reporter that faithfully reports FMRFa expression in Tv neurons. An equivalent effect on proFMRF (75%) and FMRFa-lacZ (77%) was found in sqz mutants (sqzlacZ/sqzDf and sqzGAL4/sqzDf, respectively) when compared to wild-type. Again, segment T1 is most affected with FMRFa-lacZ expressed in only 50% of T1 Tv neurons. These results show that sqz in part regulates the expression of the FMRFa gene in Tv cells (Allan, 2003).

To determine whether sqz regulates axon pathfinding of the Tv neuron, apGAL4 was used to drive the expression of a membrane-targeted reporter (UAS-EGFPF). In sqz mutants, a frequent failure of the Tv axon to innervate the DNH was observed, instead, it apparently joins the ap-fascicle. This phenotype is most pronounced within the most anterior thoracic segment (T1). In wild-type embryos, the DNH was innervated in 100% of thoracic segments, whereas sqz mutants (apGAL4/+;sqzie/sqzDf,UAS-EGFPF) show axonal innervation in 69% of T1 segment DNHs. Failure of innervation did not result from the absence of the DNH itself, since its profile was evident in affected segments. These results show that sqz is important for proper pathfinding of Tv axons and that the Tv axon often fails to diverge from the ap-fascicle in sqz mutants, apparently reverting to an 'ap-only' phenotype (Allan, 2003).

Several determinants critical for proper FMRFa expression have been identified. These include a general peptidergic cell identity, co-expression of sqz and ap, axon projection out of the VNC, and competence to respond to a retrograde signal by activating the BMP pathway. When these criteria are met, either in the endogenous or ectopic case, FMRFa expression is triggered. Importantly, none of these events are individually exclusive to the Tv cell, but they are uniquely combined in only these 6 out of the 10,000 cells in the VNC. Reconstituting this scenario in other peptidergic neurons can trigger FMRFa expression. These results are in line with the emerging theme of a critical interplay between combinatorial transcription factor codes and signal transduction pathways in regulating gene expression and provide a clear example of how these general mechanisms also apply to the specific regulation of a terminal differentiation gene in the nervous system (Allan, 2003).

Why is ectopic FMRFa expression restricted to peptidergic neurons? Conceivably, cells responding to BMP activation and sqz/ap co-misexpression may arise from precursor cells utilizing a common genetic program, resulting in a chromatin state where the FMRFa gene is accessible to activation. Currently, the lineage from which most neuropeptidergic neurons arise is unknown, and any common theme behind their generation is uncertain. FMRFa expression may also be constrained by the presence of activators common to peptidergic neurons and/or by repressors present in non-peptidergic neurons. Common properties of peptidergic neurons, such as the dense core vesicle secretory machinery and the processing of precursor peptides, may indicate the existence of common regulatory programs for all peptidergic neurons. In support of this notion, recent studies of a novel basic helix-loop-helix transcription factor, dimmed, show that this gene is specifically expressed in most if not all peptidergic neurons. In dimmed mutants, peptidergic and secretory properties of the majority of peptidergic neurons are affected, including the expression of processing enzymes and several neuropeptides, such as FMRFa. This shows that dimmed plays a key role in specifying the peptidergic fate and supports the notion of a common regulatory program for this cell type (Allan, 2003).

Previous studies found that ap is essential for axon pathfinding of the majority of ap-neurons. However, ap does not affect Tv axon pathfinding, suggesting that the role of ap in Tv cells may exclusively be to regulate FMRFa expression. In line with these results, ap mutants do not show any apparent loss of pMad accumulation in the Tv neurons. In contrast, sqz mutants have Tv axon pathfinding phenotypes, and, consequently, a partial loss of pMad staining specifically in Tv neurons. Observations of Tv axons at the midline suggest that in the absence of sqz, the Tv axon likely reverts to an 'ap-only' axonal phenotype and turns to grow along the common ap-fascicle. Given the importance of DNH innervation for FMRFa expression, axon pathfinding defects in sqz mutants likely contribute to the loss of FMRFa in some hemisegments. However, the great difference in the loss of FMRFa expression between sqz (75%) and wit (0%) argues that sqz is not critical for BMP signaling, but rather affects it indirectly by affecting Tv axon pathfinding. Moreover, the sqz axon pathfinding phenotype is only partially penetrant and fails to explain either the reduction of FMRFa expression observed in all hemisegments, or the potency of sqz (acting together with ap) to trigger ectopic expression in Va and Vap peptidergic neurons (cells whose axons already exit the VNC and are pMad-positive). Misexpression of sqz in all ap cells occasionally leads to an additional pMad/FMRFa positive cell in the ap-cluster. In these cases, no ectopic FMRFa expression is detected in any axons extending in the common ap-fascicle, only in axons projecting into the DNH. Therefore, sqz misexpression likely alters the identity of another ap-cluster cell, imposing a Tv-like axonal pathfinding behavior and causing it to ectopically innervate the DNH. Thus, it appears that sqz regulates two critical features of Tv cell identity: differential pathfinding, and FMRFa expression (both directly and indirectly) (Allan, 2003).

Why do sqz and ap function to activate FMRFa expression within only three neuropeptidergic cell types (the Tv, Va, and Vap cells) which together comprise only 18 out of ~200 peptidergic neurons in the developing Drosophila VNC? Using the specific GAL4 lines, apGAL4, VaGAL4, and VapGAL4 to drive the expression of UAS-EGFPF, it was found that all three neuronal subsets exit the VNC: Tv axons via the DNHs, Va axons via the transverse nerves, and Vap axons via the posterior A8 nerves. This observation is important in light of previous studies of tinman (tin) mutants. In tin mutants, a number of mesodermally derived tissues, including the DNHs, fail to develop. As a result, Tv axons stall at the presumptive midline exit point and, intriguingly, FMRFa expression is strongly reduced. This suggests that the DNHs may be necessary for proper FMRFa expression in Tv cells. These findings have been confirmed; in tin mutants, the DNHs are absent, and proFMRF staining is weak and only detected in 10% of Tv neurons. To address the putative target requirement for FMRFa expression in an alternative way, apGAL4 was used to express molecules that either alter Tv axon pathfinding or interfere with Tv axonal transport. roundabout (robo), a receptor that mediates repulsion from the VNC midline, was tested. In apGAL4/UAS-robo L1 larvae, Tv axons avoid the midline and fail to innervate the DNH. As predicted, this results in a loss (2%) of FMRFa-lacZ expression. Next, dominant-activated rac (UAS-racV12) was tested; it causes Tv axons to stall before reaching the midline and they fail to innervate the DNHs. This results in a complete loss (0%) of FMRFa-lacZ expression. To interfere with axonal transport, apGAL4 was used to express a dominant-negative version of the P150/Glued dynactin motor complex component (UAS-GluedDN), a molecule shown to specifically interfere with retrograde axonal transport. In apGAL4/UAS-GluedDN L1 larvae, a complete loss (0%) of FMRFa-lacZ expression was detected. Similarly, expression of the microtubule binding Tau protein, shown to interfere with axonal transport in Drosophila led to a near complete loss (4%) of FMRFa-lacZ expression (apGAL4/UAS-τ-myc). In both UAS-GluedDN and UAS-τ-myc, normal Tv axon innervation of the DNH was observed in all segments (Allan, 2003).

By co-expressing UAS-EGFPF in all scenarios outlined above, it was found that loss of FMRFa expression was not due to loss of the Tv cell, since the number of cells within the ap-cluster was unaltered in tin, UAS-robo, UAS-racV12, UAS-GluedDN, and UAS-τ-myc. Using α-Glutactin, it was found that the DNH itself is only affected in tin mutants, not in the other genotypes. Together, these results show that innervation of the DNH and retrograde signaling is essential for the expression of FMRFa (Allan, 2003).

What is the identity of the retrograde FMRFa-inducing signal? Recently, a Drosophila BMP type-II receptor, wishful thinking (wit), was implicated in mediating a retrograde signal from muscles to motor neurons, responsible for presynaptic maturation. Signaling by the TGF-β/BMP superfamily occurs via activation of a receptor complex, consisting of two type I and two type II receptors, leading to phosphorylation and nuclear translocation of a receptor Smad protein. In Drosophila, BMP signaling leads to the phosphorylation and nuclear translocation of the Smad protein Mothers against dpp (Mad), which can be monitored using antibodies specific to phosphorylated Mad (pMad) (Allan, 2003).

Using antibodies to pMad, BMP activation in peptidergic neurons was assayed. Nuclear pMad was detected not only in motor neurons, but also in the Tv, Va, and Vap neurons, demonstrating that peptidergic neurons projecting out of the VNC also show evidence of BMP activation. Accumulation of pMad in the Tv neurons commences during stage 17, immediately following DNH innervation. These results led to a test of whether Tv innervation of the DNH would be critical for pMad accumulation and consequently for FMRFa expression. Indeed, it was found that the absence of the DNH (in tin mutants), Tv axon pathfinding alterations (in apGAL4/UAS-robo and apGAL4/UAS-racV12) and interference with Tv axonal transport (in apGAL4/UAS-GluedDN and apGAL4/UAS-τ-myc) are all accompanied by loss of pMad staining specifically in Tv neurons. The ectopic ap-cluster FMRFa-expressing cell induced by sqz misexpression is also pMad positive. Given the role of sqz in Tv axon pathfinding, this is interpreted as resulting from sqz dominantly altering the projection of one other ap-cluster cell, forcing it to innervate the DNH. Thus, in all genotypes examined, Tv axonal projection to the DNH is critical for pMad accumulation (Allan, 2003).

Since Wit is expressed in a restricted pattern in the developing VNC, attempts were made to address whether the Tv neurons express Wit. However, single-cell resolution could not be obtained with the Wit antibody and Wit could not be definitely localized in Tv cells. However, the wit-dependent pMad accumulation in Tv neurons, the apGAL4/UAS-tkvA, UAS-saxA-mediated rescue of wit mutants, and the UAS-gbb-mediated 'rescue' of UAS-robo misexpression, provide genetic evidence supporting the expression of wit in Tv cells. Previous studies have shown that gbb is expressed in developing endoderm and visceral mesoderm, but it has not been detected in the VNC. By in situ hybridization, no apparent expression was detected in the DNH. Given that the DNH only contains two cell bodies, low-level gbb expression may be beyond detection. Moreover, since the anterior midgut is positioned in very close proximity to the DNHs, it is possible that Gbb diffuses from the visceral mesoderm to the DNH (Allan, 2003).

Why is BMP activation necessary for FMRFa expression? Neither forced axonal exit from the VNC (apGAL4/UAS-Unc5) nor autocrine presentation of the Gbb ligand (apGAL4/UAS-gbb) leads to activated pMad and FMRFa expression in ap cells other than the Tv cell. This indicates that the Tv cell is uniquely predetermined to respond to the Gbb ligand. In fact, even direct activation of the BMP pathway (UAS-saxA, -tkvA;apGAL4/+) in all ap neurons does not trigger ectopic FMRFa expression, showing that the Tv cell is further uniquely capable of responding to BMP activation. The misexpression results show that both of these properties of the Tv cell are specified by sqz/ap co-expression. Given this level of Tv cell predetermination, it begs the question as to why Tv cell FMRFa expression evolved to be dependent upon a retrograde BMP signal. Perhaps dependence upon a retrograde signal provides precise control over the onset of FMRFa expression during embryogenesis. In fact, Tv neurons are born by stage 14 (as evident by ap expression) but do not activate FMRFa expression until late stage 17, upon DNH innervation. Alternatively, the presence of a small number of sqz/ap co-expressing cells in the developing brain that do not express FMRFa may necessitate additional regulatory control over FMRFa expression. Dependence upon a signal transduction pathway also provides several unique means of control and amplification of target gene expression. Finally, the fact that sqz, ap, and BMP activation only act to trigger FMRFa expression within a neuropeptidergic cellular context reveals additional complexity underlying the control of specific neuropeptide expression. Given the large number of diverse cell types in the CNS, what may appear to be an almost excessive complexity of combinatorial coding may in fact be essential for high fidelity of gene expression (Allan, 2003).

Protein Interactions

Nab proteins form an evolutionarily conserved family of transcriptional co-regulators implicated in multiple developmental events in various organisms. They lack DNA-binding domains and act by associating with other transcription factors, but their precise roles in development are not known. This study analyzed the role of nab in Drosophila development. By employing genetic approaches it was found that nab is required for proximodistal patterning of the wing imaginal disc and also for determining specific neuronal fates in the embryonic CNS. Two partners of Nab were identified: the zinc-finger transcription factors Rotund and Squeeze. Nab is co-expressed with squeeze in a subset of neurons in the embryonic ventral nerve cord and with rotund in a circular domain of the distal-most area of the wing disc. These results indicate that Nab is a co-activator of Squeeze and is required to limit the number of neurons that express the LIM-homeodomain gene apterous and to specify Tv neuronal fate. Conversely, Nab is a co-repressor of Rotund in wing development and is required to limit the expression of wingless (wg) in the wing hinge, where wg plays a mitogenic role. Pull-down assays show that Nab binds directly to Rotund and Squeeze via its conserved C-terminal domain. Two mechanisms are described by which the activation of wg expression by Rotund in the wing hinge is repressed in the distal wing (Félix, 2007).

Precise temporal and spatial control of gene transcription is crucial for development. Sequence-specific DNA-binding factors and their association with a variety of modulator proteins, the co-factors, achieve this control. Co-factors do not bind DNA but act as adaptors between DNA-binding factors and other proteins. A number of transcription factors have been characterized, many of which act by recruiting multiprotein complexes with chromatin-modifying activities. By recruiting co-factors, a DNA-binding protein can act as co-activator or as co-repressor depending on the context. An example of a co-repressor is the retinoblastoma protein that converts the E2F transcription factor into a repressor of cell-cycle genes. The identification of co-factors and the determination of their precise roles are crucial for understanding the mechanisms that govern development (Félix, 2007).

Nab (NGFI-A-binding protein) proteins form an evolutionarily conserved family of transcriptional regulators. Nab was originally identified in mouse as a strong co-repressor by virtue of its capacity to interact directly with the Cys2-His2 zinc-finger transcription factor Egr1 (Krox24; NGFI-A) and inhibit its activity. Two Nab genes, Nab1 and Nab2, have been identified in vertebrates. Nab proteins do not bind DNA but they can repress (Svaren, 1998) or activate (Sevetson, 2000) gene expression by interacting with Egr transcription factors. Nab proteins have two regions of strong homology: NCD1 and NCD2. The NCD1 domain interacts with the R1 domain of Egr1 (Svaren, 1998). The NCD2 domain is required for transcriptional regulation (Swirnoff, 1998). Mice harboring targeted deletions of Nab1 and Nab2 have phenotypes very similar to Egr2 (Krox20)-deficient mice, suggesting that they act as co-activators of this gene (Le, 2005). In zebrafish, egr2 controls expression of the Nab gene homologs in the r3 and r5 rhombomeres of the developing hindbrain (Mechta-Grigoriou, 2000). Egr2 has been implicated in determining the segmental identities of r3 and r5 by controlling the expression of several target genes as well as cell proliferation. Misexpression experiments suggest that Nab1/Nab2 antagonize Egr2 transcriptional activity by a negative-feedback regulatory loop. Nevertheless, Nab proteins might have additional functions as these experiments also led to alterations of the neural tube not found in Egr2-deficient embryos (Mechta-Grigoriou, 2000). Conversely, Egr2-deficient mice have a severe hindbrain segmentation defect that is not found in mice deficient in Nab1 and Nab2. Nab might also have Egr-independent functions in mice because, although epidermal hyperplasia has been observed in Nab1 Nab2 double mutant mice, this phenotype has not been observed in mice lacking any of the Egr proteins (Le, 2005; Félix, 2007 and references therein).

In Drosophila, only one Nab gene has been identified; it is highly homologous to vertebrate Nab genes in the NCD1 and NCD2 domains. Drosophila nab mutants are early larval lethal. Detection of nab transcripts by in situ hybridization indicates expression in a subset of neuroblasts of the embryonic and larval CNS and weak expression in imaginal discs (Clements, 2003). The role of Nab in Drosophila development is not known and so far no binding partner has been identified. This report shows that nab is a component of the combinatorial code that determines the number of neurons that express the gene apterous (ap) in embryonic neural development, and that nab specifies the Tv neuronal fate in the ap thoracic cluster of neurons (Félix, 2007).

In early larval development, the wing fate is established in the distal-most region of the wing disc by a combination of two factors: activation of the gene vestigial (vg) and repression of the gene teashirt (tsh). Later, in early third instar larvae, wingless (wg) is activated in a ring of cells (the inner ring, IR) that borders the vg expression domain in the presumptive wing region. It has been suggested that activation of the IR involves a signal from the vg-expressing cells to the adjacent cells. Interpretation of this signal by the adjacent cells requires the transcription factors encoded by rotund (rn) and nubbin (nub). Expression of wg in the IR plays a mitogenic role; hence, as a consequence of wg expression, cells proliferate and the IR moves away from the vg border. At a distance from the source of the signal that drives the initial activation, wg IR expression is maintained by an autoregulatory loop that involves homothorax (hth). It is thought that an additional mechanism distally represses wg IR expression and, in so doing, controls cell proliferation in the wing hinge. In this report, it is shown that during imaginal disc development, nab is strongly expressed in the wing presumptive domain under the control of vg, and that nab is required in proximodistal axis development to control the expression of wg in the wing hinge (Félix, 2007).

Two putative partners of Nab have been identified: Rn and Squeeze (Sqz). These proteins are members of the Krüppel family of zinc-finger proteins. Pull-down assays show that that Nab interacts with both proteins via a conserved C-terminal domain, and evidence is presented that Nab acts as co-activator of Sqz in embryo development and as co-repressor of Rn in wing development. Finally, it is proposed that there are two mechanisms to repress the activation of wg expression by Rn in the wing pouch: the first involves Nab as a co-repressor of Rn; the second involves Sqz as a competitor of Rn for binding to specific DNA target sites (Félix, 2007).

Expression of Nab

Antibody against Nab revealed a low level of expression in all imaginal discs. In late third instar wing discs, Nab was strongly expressed in a circular domain that delimits the expression of wg in the inner ring. Nab expression was first detected in early third instar larvae, in a group of cells of the distal-most wing, and was maintained throughout the remainder of the larval and pupal stages. There was a low level of expression in the rest of the wing disc, except in the hinge where there was no detectable expression. In the eye disc, Nab was detected in a stripe corresponding to the morphogenetic furrow (Félix, 2007).

The Rn zinc-finger transcription factor is a potential partner of Nab in wing development

The mammalian Nab partner Egr1 contains an inhibitory domain called R1. When this domain is deleted the transcriptional activity of Egr1 increases 15-fold. It has been shown that the R1 domain mediates a functional interaction between Nab and Egr1. Since no R1 domain has been identified in the fly genome and all the previously identified partners of Nab are Krüppel-type zinc-finger transcription factors, transcription factors of the Krüppel family expressed in the wing were examined as potential Nab partners in the fly. The gene rn encodes a Krüppel-like zinc-finger protein that in the wing is expressed in a circular domain slightly broader than the nab domain. The wg inner ring enhancer is active only in the cells that express rn and that do not express nab. Previous studies have shown that Rn is required for activation of the wingless spd enhancer. The results so far suggest that Rn could be a partner of Nab in the wing: first, nab is expressed in the rn-expressing cells that do not express wg; second, nab loss-of-function clones contain ectopic Wg; and third, nab misexpression represses the wg inner ring enhancer (Félix, 2007).

rn was also expressed in leg discs in a broad ring that corresponded to three tarsal segments (T2-4). In rn mutant legs, the T2-4 tarsal segments were deleted. It would therefore be expected that if Rn were a partner of Nab, ectopic expression of nab in the leg would generate the same phenotype as the lack of Rn. This proved to be the case when nab was misexpressed in the rn expression domain under the control of the rnGal4 driver. The phenotype of these flies was indistinguishable from the rn mutant phenotype in both legs and wings. The specificity of this interaction was examined by rescuing the phenotype caused by nab misexpression by co-expressing rn (rnGal4>UASrn+UASnab), as well as by misexpressing nab in a broader domain using Distal-less Gal4 (DllGal4), which is expressed from mid-tibia to distal leg (DllGal4>UASrn). In the first experiment, the phenotype was markedly reduced in both wing and leg, indicating that adding more rn antagonizes the inhibitory effect of nab misexpression. In the second experiment, although nab was misexpressed in a broader domain of the leg, the phenotype was unaltered and was restricted to the area where rn was expressed. Taken together, these results support a role for Rn as a potential partner of Nab and that Nab acts as co-repressor of Rn function in the cells where both are expressed. The rn mutant phenotype in the wing is caused by the loss of wg expression in the inner ring. Whether wg expression was affected in rnGal4 UASnab and rnGal4 UASnab UASrn wings was examined. In the first case, the inner ring was found to be absent, whereas in the second it was partially restored. In summary, these results indicate that Nab functions in wing development by antagonizing the transcriptional activation function of Rn (Félix, 2007).

The Sqz zinc-finger transcription factor is a potential partner of Nab in neuronal fate specification

Although nab loss-of-function alleles are larval lethal, the rn-null condition is homozygous viable. This suggests that Nab may have at least one other partner in embryonic development. Rn belongs to a conserved subfamily of zinc-finger proteins that include Drosophila Sqz, C. elegans LIN-28 and rat Ciz. Sqz and Rn have two highly homologous domains: the zinc-finger domain (90% identity) and a 32 amino acid C-terminal domain (over 80% identity). sqz mutant alleles are larval lethal and have a motility defect. sqz is first required in embryonic CNS development to define the number of cells that express the LIM-homeodomain gene ap in the ap thoracic cluster of interneurons. Later on, it is also involved in the combinatorial code of transcription factors that specifies the fate of the Tv neuron in the ap cluster. The Tv neuron is distinguished from the rest of the neurons in the cluster by the fact that it contains the neuropeptide FMRFa [FMRFamide-related (Fmrf)]. In sqz mutant embryos, additional ap-expressing neurons are generated and the Tv neuron is not specified as no FMRFa expression is found (Allan, 2003). To determine whether Nab is a co-factor of Sqz, the expression of nab and sqz was examined in stage-17 embryos. It was found that a subset of the CNS neurons that express sqz also expressed nab, whereas other neurons express either sqz or nab. Two or three neurons in the ap cluster of stage-17 embryos express nab, one typically at a relatively high level of expression. By the first instar larval stage only one neuron in the ap cluster expressed nab. By double staining with anti-FMRFa and anti-Nab it was possible to identify this as the Tv neuron. At this stage, sqz was expressed at high levels in the Tv neuron and at low levels in two other neurons of the ap cluster. Next the expression of ap and FMRFa was analyzed in nab mutant larvae. In first instar nabSH143 larvae, additional ap-expressing neurons were found in the ap cluster. In nabSH143 embryos, additional cells expressed the bHLH gene dimmed (dimm), as shown for sqz mutants. The expression of FMRFa was analyzed in the ap clusters of first instar larvae, and FMRFa staining was lost or reduced in all the Tv neurons, mainly in the T1 cluster. It is concluded that lack-of-function alleles of nab and sqz generate the same embryonic phenotypes: the number of ap-expressing cells in the ap thoracic clusters is increased, additional dimm-expressing neurons are detected in the clusters, and Tv neuronal fate is absent. These results strongly suggest that, unlike the situation in imaginal disc development where Nab acts as a co-repressor of Rn, in CNS development Nab is required as a co-activator of Sqz (Félix, 2007).

Nab binds directly to Rn and Sqz via a conserved C-terminal domain

In order to analyze the molecular role of Nab as a co-factor of Sqz and Rn GST pull-down assays were performed. The complete nab cDNA was cloned in a glutathione S-transferase (GST) vector and incubated with radioactively labeled Rn or Sqz. Nab-GST, but not GST alone, readily retained [35S]methionine-labeled Rn or Sqz. Rn and Sqz share a C-terminal domain of 32 amino acids with a homology greater than 80%. To further test whether this domain mediates the interaction with Nab, the pull-down assays were repeated with an [35S]Rn in which the C-terminal domain was deleted. This deletion removes the region from amino acid 894 to the C-terminus (943) of the protein (RnΔ894). The ability of Nab-GST to retain the [35S]RnΔ894 was notably reduced. It is concluded that this conserved domain mediates the direct interaction of Nab with Rn and Sqz. To further test whether the C-terminal domain is sufficient to mediate this interaction, the Nab-GST was incubated with a 32 amino acid peptide containing just the sequence of the C-terminal domain. Nab-GST did not retain the peptide, indicating that the C-terminal domain is not sufficient to mediate Nab-Rn interaction. Since no other conserved domains have been identified between Rn and Sqz besides the zinc-finger and C-terminal domains, it is considered that either secondary structure or an additional modification of the protein is required for binding Nab. In order to provide an in vivo functional test of this hypothesis, the rnΔ894 fragment was cloned into the pUAST vector and clones of cells misexpressing UASrnΔ894 were generated (Act>Gal4>UASrnΔ894). These clones activated the expression of wg throughout the wing pouch. As a control experiment, the wild-type version of rn (Act>Gal4>UASrn) was misexpressed. These clones only activated wg expression in the wing hinge, outside of the nab expression domain (Félix, 2007).

Sqz competes with Rn in wing disc development

sqz expression was examined in the wing disc. Because of the high degree of sequence homology between rn and sqz and to avoid interference with the rn mRNA present in the wing, in situ hybridization assay was performed in rn mutant discs. sqz expression was detected by in situ hybridization in rn20 wing discs in a circular pattern that faded off laterally and whose proximal limit coincided with the limit of vg expression; this corresponded to the distal-most wing fold. To determine whether sqz plays a role in wing development the phenotype was analyzed of sqz mutant clones induced by mitotic recombination. These clones had no adult phenotype, nor did they alter the expression of wg. Since Sqz and Rn share zinc-finger and the C-terminal domains and differ in their N-terminal domains, it was asked whether the roles of Sqz and Nab might be functionally redundant, both repressing Rn activity but by different mechanisms: Nab would repress Rn activity by direct binding to Rn protein as a co-repressor, whereas Sqz would compete for binding to the same DNA targets. To test this hypothesis, the effect was analyzed of misexpressing sqz in the rn expression domain. rnGal4/UASsqz UASGFP flies had small deletions of the wing hinge and shortened legs, a phenotype that resembles the nab misexpression and rn mutant phenotypes. In agreement with these results, wg expression in the inner ring was downregulated in rnGal4/UASsqz wing discs. An alternative explanation for these results is that sqz activates nab expression, but no nab misexpression was seen in this experiment. It is suggested that there must be some functional redundancy, irrespective of whether Nab and Sqz play similar roles in the wing by repressing Rn activity, and this would account for the low penetrance of the nab mutant clones. Because nab and sqz map on different chromosome arms it was not possible to generate double-mutant clones. Therefore nabSH143 homozygous clones were generated in a sqzlacZ/+ background. In this situation, the frequency of clones misexpressing wg increased by 38%). It was also noted that the clones that showed wg misexpression were preferentially located in the lateral-most regions of the wing, which correspond to the regions with the lowest levels of sqz expression. Taken together, these observations support the hypothesis that Nab and Sqz play similar roles in wing development: Nab as a co-repressor of Rn via its conserved C-terminal domain, and Sqz by competing with Rn for binding to its DNA targets. This function of Sqz would differ from its above-proposed role as a transcriptional activator in CNS development, and would not require Nab (Félix, 2007).

This study presented evidence that Nab is a co-activator of Sqz. This protein has been implicated in two aspects of embryonic ventral nerve cord development: first, in a Notch-dependent lateral inhibition mechanism that specifies the number of cells that express ap in the ap thoracic neuronal cluster; and second, in the specification of the Tv neuronal fate. nab and sqz are co-expressed in a subset of neurons, including several of the ap cluster, as well as the Tv neuron. nab loss-of-function embryos reproduce all the phenotypes of sqz loss-of-function embryos: additional cells express ap in the cluster and the Tv neuronal fate is lost. In addition, in both nab and sqz mutants an increased number of cells in the clusters express dimm. These findings indicate that Nab is required for all identified Sqz functions in embryonic development. Although this analysis focused on the ap thoracic cluster of neurons, both sqz and nab are co-expressed in many cells in the ventral nerve cord and others expressed either sqz or nab. But no other functions have been identified for sqz and it is not known how the expression of sqz is controlled. It has been reported that the expression of nab in the ventral nerve cord depends on the gene castor (Clements, 2003). Thus, the results presented in this study reveal greater complexity in the mechanisms of neuronal fate specification. The combined expression of genes, whose expression is individually activated by different mechanisms, is required to determine specific neuronal fates (Félix, 2007).

Sqz and Rn share two regions of strong homology: the zinc finger and a stretch of 32 amino acids in the C-terminal domain. By contrast, only rn has a long N-terminal domain. The results indicate that the C-terminal domain mediates the interaction with Nab. By GST pull-down assays, it was shown that Nab binds to the full-length Rn protein but not to the RnΔ894 version, and clones of cells misexpressing rnΔ894 activate wg expression in the nab expression domain. The similarity between sqz misexpression and rn loss-of-function phenotypes in leg and wing suggests that Sqz acts like a dominant-negative form of Rn in the rn domain: both proteins would bind to the same target sites but have opposite effects, and the results indicate that this role of Sqz would not require interaction with Nab. It is possible that the long N-terminal region of Rn is involved in interaction with other partners specifically required for Rn function (Félix, 2007).

Thus, these results indicate that Nab has a dual role as co-repressor of Rn and co-activator of Sqz. Previous studies in vertebrates also suggest that Nab is involved in both repression and activation of transcription. Co-repressors are proteins that bridge the interaction of the repressor with its target. Two main co-repressors have been identified in Drosophila: Groucho and CtBP. CtBP binds to a specific sequence motif (P-DLS-K) that has been found in the sequence of three repressors present in the early embryo: Snail, Knirps and Krüppel. All three are zinc-finger transcription factors, and genetic evidence suggests that they all require CtBP to repress their targets. Neither Rn nor Sqz have a CtBP-binding motif but one has been in Nab (P-DLS--K). Although the functional significance of this motif remains to be confirmed, itis suggested that Nab is acting as a bridge between Rn and CtBP (Félix, 2007).


DEVELOPMENTAL BIOLOGY

Embryonic

The expression of sqz is largely restricted to subsets of cells in the CNS throughout embryonic and first instar larval (L1) development. Using sqzGAL4 to drive expression of the axonal reporter, UAS-τ-myc, sqz was found to be expressed in a population of lateral interneurons, primarily projecting axons in the anterior and posterior commissures. In sqz mutants, expressing neurons are generated and appear to extend axons along the appropriate tracts. Using both sqzlacZ and sqzGAL4, tests were performed for overlap with ap; sqz and ap were found to be co-expressed specifically within the thoracic ap cluster. Co-expression of sqz and ap is evident from the onset of ap expression at stage 14, with one neuron typically expressing higher levels of sqz. By stage 17, sqz expression is restricted to two neurons within the ap-cluster, with one neuron typically continuing to display higher levels of expression. Expression overlap between sqz and FMRFa was tested in late stage 17 embryos, when FMRFa expression commences; sqz is indeed selectively expressed at higher levels within the FMRFa Tv neuron. Thus, the six neurons within the VNC that co-express ap and higher levels of sqz selectively express the neuropeptide FMRFa and innervate the three specialized neuroendocrine glands -- the dorsal neurohemal organs (Allan, 2003).


EFFECTS OF MUTATION
CG5557 has a larval lethal phase. Mutants eclose at a low frequency as immotile adults that die within 24 hr. Mutant larvae display a motility defect whereby the body wall musculature over-contract radially during the peristaltic wave typical of insect larval motility, apparent as a 'squeezing' of the intestine. Since this motility phenotype is fully penetrant and scored with 100% accuracy (sqzlacZ/sqzDf), CG5557 was renamed squeeze (sqz) (Allan, 2003).

Regulators acting in combinatorial codes also act independently in single differentiating neurons

In the Drosophila ventral nerve cord, a small number of neurons express the LIM-homeodomain gene apterous (ap). These ap neurons can be subdivided based upon axon pathfinding and their expression of neuropeptidergic markers. ap, the zinc finger gene squeeze, the bHLH gene dimmed, and the BMP pathway are all required for proper specification of these cells. Here, using several ap neuron terminal differentiation markers, how each of these factors contributes to ap neuron diversity has been resolved. These factors interact genetically and biochemically in subtype-specific combinatorial codes to determine certain defining aspects of ap neuron subtype identity. However, it was also found that ap, dimmed, and squeeze additionally act independently of one another to specify certain other defining aspects of ap neuron subtype identity. Therefore, within single neurons, single regulators acting in numerous molecular contexts differentially specify multiple subtype-specific traits (Allan, 2005).

Within every VNC hemisegment, ap is expressed by one dorsal neuron (dAp) and two ventral neurons (vAp). Additionally, in thoracic VNC hemisegments, ap is expressed by a lateral cluster of four neurons (the ap cluster), termed the Tv, Tvb, Tva, and Tvc neurons. These ap neurons are phenotypically diverse. The axons of most ap neurons project within an ipsilateral fascicle (ap fascicle) that projects to the brain, whereas the axons of the Tv cell exit the VNC at the midline to innervate the dorsal neurohemal organs (DNH). A subset of ap neurons is peptidergic (the Tv, Tvb, and dAp neurons). As is characteristic for the vast majority of Drosophila peptidergic neurons, these cells express high levels of the peptide biosynthetic enzyme peptidylglycine alpha-hydroxylating monooxygenase (PHM). However, this peptidergic subset is also diverse: Tv cells selectively express the dFMRFa neuropeptide, whereas Tvb and dAp cells selectively coexpress three peptide biosynthetic enzymes -- PC2, Furin1, and PAL2 -- although the identity of their secreted neuropeptide(s) remains unknown. This coexpression in Tvb and dAp cells suggested a functional grouping and a common name, 'Ap-let' cells. For clarity, the ap neurons will be considered as three classes: (1) Tv cells express dFMRFa and PHM and innervate the DNH; (2) Ap-let (Tvb and dAp) cells express PHM, PC2, Furin1, and PAL2; (3) the vAp, Tva, and Tvc cells are nonpeptidergic (Allan, 2005).

ap, sqz, dimm, and the BMP pathway act in a combinatorial code to regulate dFMRFa in the Tv cell (ap, sqz, dimm, and the BMP pathway) and furin1 (ap, dimm) in Ap-let cells. Importantly, however, each regulator also plays critical roles within these ap neurons independent of the other regulators. Ap independently acts to regulate axon pathfinding by all ap cells except the Tv. Dimm independently controls PHM in the Tv and Ap-let cells. Moreover, Sqz independently acts via the N pathway to regulate cell identity within the ap cluster, upstream of both Ap and Dimm, apparently by suppressing the Tvb cell fate to favor the Tv fate. The Ap-let cells do not express Sqz, nor do they have an activated BMP pathway. In these neurons, Ap activates the expression of Dimm, and both act together to activate the expression of the peptide-processing enzyme Fur1. The Tva and Tvc cells of the ap cluster do not express Dimm and do not have an activated BMP pathway. Remarkably, the differences inferred between regulatory circuits for the two classes of peptidergic cells are highly reminiscent of differences in regulatory circuits that operate during the differentiation of distinct noradrenergic neurons. Together, these sets of studies support the proposition that epistatic relations between regulators underlying the production of a common phenotype may differ according to cell type (Allan, 2005).

The loss-of-function and gain-of-function phenotypes presented for ap, sqz, dimm, and the BMP pathway, suggest that they act in a combinatorial fashion to regulate dFMRFa expression in the Tv neuron. Likewise, the results indicate that ap and dimm, in the absence of sqz and the BMP pathway, combine to activate Fur1 in the Ap-let neurons, Tvb and dAp. In order to determine whether these regulators act simultaneously on dFMRFa and Fur1, rather than in a genetic hierarchy, the epistatic and biochemical relationship between these regulators were studied. Only one clear epistatic relationship was found; Ap activates the expression of Dimm in the majority of ap neurons. Therefore, it was important to determine whether Dimm acted downstream of Ap to independently and more directly regulate dFMRFa and Fur1 expression. This hypothesis was tested in two complementary tests. (1) Rescuing Dimm function in ap neurons that were absent for Ap function, yielded a nearly complete rescue of dFMRFa in Tv neurons, but only relatively weak rescue of Fur1 in Ap-let neurons. (2) Panneuronal co-misexpression of both ap and dimm triggers ectopic dFMRFa expression in a much greater number of neurons than does either regulator alone. These data indicate that Dimm functions together with Ap to achieve wild-type levels of dFMRFa and, more notably, Fur1. Thus, ap and dimm appear to display both hierarchical and combinatorial interactions. This hypothesis has precedent in studies of the developing pancreas, in which Foxa2 is required for pdx-1 transcription in β cells and later interacts directly with PDX-1 protein to regulate target gene expression, including maintained pdx-1 expression. Biochemical data are also consistent with the possibility that a combinatorial Ap, Dimm, and Sqz code that activates dFMRFa and dFur1 involves direct protein interactions. These may exist within larger complexes bridged by Chip, since Dimm can interact directly with both Ap and Chip, and this in turn may explain why Dimm partially rescues both the ap mutant dFMRFa and Fur1 phenotypes. These multiple interactions are reminiscent of synergistic interactions suggested between mammalian bHLH proteins, LIM-HD proteins, and the Chip homolog, LDB1/NLI. The simplest explanation for restricted patterns of neuropeptides and certain neuropeptide biosynthetic enzymes features a combinatorial hypothesis. More specifically, it is proposed that different combinatorial codes of transcription factors act cell specifically to effect differing patterns of neuropeptides and associated processing enzymes (Allan, 2005).

Ap expression is an early marker of ap cell differentiation, and it is required for proper axonal pathfinding by most ap neurons, although not by the Tv cell. In contrast, neither Sqz nor Dimm appear to control ap cell morphogenesis. An independent role for Sqz occurs early in ap cell differentiation, at a time when postmitotic cell fates are being determined. It is surprising that such cell fate changes can be rescued by UAS-Dl. Why would the frequently used N pathway signaling system depend upon a much more restricted regulator like sqz for proper activity? Increasing evidence points to major mechanistic differences between N signaling during neuroblast specification and during asymmetric division, where asymmetric divisions specifically require neuralized, numb, and sanpodo. No expression of sqz is found in neuroblasts, but expression is evident in many VNC cells. Therefore, it is proposed that factors like Sqz coordinate late N signaling with cell specification and/or cell cycle genes (Allan, 2005).

Dimm acts independently of Ap, Sqz, and the BMP pathway to activate expression of the neuropeptide-processing enzyme PHM. The evidence regarding the independent role of Dimm suggests that it is a master regulator of neuroendocrine cell fate. dimm expression is highly correlated with a neuroendocrine/peptidergic cellular identity, where it regulates the expression of almost all neuropeptides and their processing enzymes examined to date, especially within those neurons that express peptides that are processed to include an α-amidated C terminus. This is a significant cellular pattern, because more than 90% of Drosophila neuropeptides are amidated. Furthermore, high-level expression of the PHM enzyme is absolutely required for amidation and serves as an excellent marker for most peptidergic neurons in Drosophila. Finally, PHM expression appears to be dedicated to neuroendocrine peptide biosynthesis; it is exclusively found within the luminal domain of secretory vesicles. Thus, PHM expression provides a faithful marker for the peptidergic/neuroendocrine cell fate. This study has shown that PHM is dominantly induced by dimm overexpression throughout most or all of the CNS. This evidence, together with the loss-of-function data argues strongly that dimm is a neuroendocrine master regulator, with properties akin to those of other bHLH proteins in regulating cell fate (Allan, 2005).

As anticipated, more restricted peptidergic traits such as dFMRFa and Fur1 expression are dependent upon combinatorial codes. Importantly, however, the selection of cell-specific peptidergic markers arises from a deterministic interaction between a peptidergic master regulator and a cell-specific combinatorial code. There exists a clear analogy between the action of dimm in developing neurons and results regarding the glial cells missing (gcm) gene. Studies have shown that gcm is both necessary and sufficient for glial cell specification within the DrosophilaVNC. gcm is able to ectopically activate generic glial genes, such as reversed polarity, and also activates subclass-specific glial genes, but only in certain prescribed subsets of cells. Thus, similar to gcm, it is predicted that dimm is a master regulator of core neuroendocrine genes in most peptidergic/neuroendocrine cells. It will be of interest to determine which genes beyond PHM are under dimm control. In parallel, dimm combines with local-acting factors to help activate subclass-specific genes (e.g., neuropeptide-encoding genes) within peptidergic cell subsets (Allan, 2005).

The genes studied here combine to regulate dFMRFa and Fur1 but also have independent roles within the same cells. This raises the issue of how Dimm, for instance, can complex with Ap/Sqz on dFMRFa and also act independently on PHM within the same nucleus. Surprisingly, no clear evidence of an antagonistic relationship between the individual roles of Ap, Sqz, and Dimm was found. For example, co-misexpression of ap with dimm does not obviously suppress the ectopic PHM expression observed when dimm alone is misexpressed. Likewise, misexpression of sqz in the Fur1-expressing dAp/Tvb cells does not suppress Fur1. Thus, it appears that the independent mechanisms of regulator action are robust and can coexist with combinatorial functions. Therefore, it is proposed that these regulators operate within a bistable organizational mechanism. With respect to independent roles, it is proposed that Dimm operates independently of Ap and Sqz to dominantly induce specific target genes (e.g., PHM) within all neuronal lineages by forming heterodimers with a class A bHLH like Da, or by forming homodimers. The Drosophila bHLH Twist protein has distinct regulatory roles in vivo, acting either as a heterodimer with Da, or as a homodimer. Notably, the mammalian ortholog of Dimm, Mist1, forms functional homodimers to promote the differentiation of pancreatic secretory cells (Allan, 2005).

The TGFβ/BMP signal transduction pathway plays critical roles during a number of developmental events, and mutants affecting the Drosophila BMP pathway show dramatic defects in embryonic development. In contrast, in the Tv neuron, BMP signaling plays a much more subtle role, and although it is critical for dFMRFa expression, no effects were found upon the expression of sqz, ap, or dimm or on the general peptidergic marker PHM in wit mutants. Although these studies cannot rule out other roles for the BMP pathway in Tv neurons, it is tempting to speculate that target-derived BMP signaling in neurons may have quite a limited set of nuclear readouts in each specific neuronal subclass (Allan, 2005).

Intrinsic control of precise dendritic targeting by an ensemble of transcription factors

Proper information processing in neural circuits requires establishment of specific connections between pre- and post-synaptic neurons. Targeting specificity of neurons is instructed by cell-surface receptors on the growth cones of axons and dendrites, which confer responses to external guidance cues. Expression of cell-surface receptors is in turn regulated by neuron-intrinsic transcriptional programs. In the Drosophila olfactory system, each projection neuron (PN) achieves precise dendritic targeting to one of 50 glomeruli in the antennal lobe. PN dendritic targeting is specified by lineage and birth order, and their initial targeting occurs prior to contact with axons of their presynaptic partners, olfactory receptor neurons. A search was performed for transcription factors (TFs) that control PN-intrinsic mechanisms of dendritic targeting. Two POU-domain TFs, acj6 and drifter have been identified as essential players. After testing 13 additional candidates, four TFs were identified, (LIM-homeodomain TFs islet and lim1, the homeodomain TF cut, and the zinc-finger TF squeeze) and the LIM cofactor Chip, that are required for PN dendritic targeting. These results begin to provide insights into the global strategy of how an ensemble of TFs regulates wiring specificity of a large number of neurons constituting a neural circuit (Komiyama, 2007).

Neuroblast entry into quiescence is regulated intrinsically by the combined action of spatial Hox proteins and temporal identity factors.

Neural stem cell quiescence is an important feature in invertebrate and mammalian central nervous system development, yet little is known about the mechanisms regulating entry into quiescence, maintenance of cell fate during quiescence, and exit from quiescence. Drosophila neural stem cells (neuroblasts or NBs) provide an excellent model system for investigating these issues. Drosophila NBs enter quiescence at the end of embryogenesis and resume proliferation during larval stages; however, no single neuroblast lineage has been traced from embryo into larval stages. This study establishes a model NB lineage, NB3-3, which allows reproducibly observation of lineage development from NB formation in the embryo, through quiescence, to the resumption of proliferation in larval stages. Using this new model lineage, a continuous sequence of temporal changes is shown in the NB, defined by known and novel temporal identity factors, running from embryonic through larval stages; quiescence suspends but does not alter the order of neuroblast temporal gene expression. NB entry into quiescence is regulated intrinsically by two independent controls: spatial control by the Hox proteins Antp and Abd-A, and temporal control by previously identified temporal transcription factors and the transcription co-factor Nab (Tsuji, 2008).

This study has revealed for the first time the temporal changes in a Drosophila NB lineage from embryonic NB formation, through entry into quiescence, to resumption of proliferation in larval stages. Using a model NB system with which the complete lineage formation can be reproducibly traced at the resolution of individual cell divisions, it was shown that despite considerable differences in extracellular environment the temporal changes (as defined by the switching of transcription factor/co-factor expression) proceeded continuously in each NB throughout the embryonic and larval stages. Moreover, mutual regulation was found between quiescence and the series of the temporal transcription factors/co-factor; the temporal transcription factors/co-factor endogenously control the timing of triggering NB quiescence, which in turn suspends the switching of late temporal transcription factor expression (Tsuji, 2008).

In the Antp mutant and following ectopic expression of Abd-A there was a lack of NB quiescence, and consequently what appeared to be a precocious generation of larval neurons during embryogenesis was observed. This strongly supports the notion that temporal changes in NBs actually continue in sequence before and after quiescence, i.e., through embryogenesis and larval stages, and in the absence of quiescence the changes occur precociously. In addition, this indicates that spatial and temporal factors control NB quiescence through independent routes (Tsuji, 2008).

Antp mutants did not exhibit NB3-3T quiescence in all thoracic T1-T3 segments. In Antp mutants, epidermis in T2 and T3 segments transform into that in the T1 segment, and some thoracic NB lineages retain thoracic-specific features. These facts indicate that the inhibition of NB3-3T quiescence by Antp mutation is not just a consequence of global transformation of thoracic-to-abdominal segments but rather results from specific effects on individual NBs. NB-specific misexpression of Abd-A also repressed Antp and inhibited NB3-3T quiescence. This also provides evidence that the effect is specific to NBs. Furthermore, because the effect could be observed even when Abd-A was induced after several divisions of the NB, thoracic NBs probably maintain plasticity of their identities during lineage formation (Tsuji, 2008).

It was shown that the temporal transcription factors/co-factor Pdm, Cas, Sqz and Nab play a role in triggering NB quiescence intrinsically in NBs. All of these factors also controlled temporal specification within late lineages of embryonic NBs in both thoracic and abdominal segments. This was confirmed by further examining the relationships of the temporal factors. For example, the loss of Pdm function in NB3-3T resulted in precocious transcription factor switching and precocious quiescence. Conversely, in cas mutant embryos, in which Pdm expression was de-repressed, quiescence was inhibited and expression of late-stage-specific temporal factors disappeared. Similar to Pdm upregulation, loss of nab function resulted in loss of both transcription factor switching and quiescence (Tsuji, 2008).

Although Nab and Sqz can form a complex, nab and sqz mutants displayed very different phenotypes. Both mutants showed de-repression of Kr expression; however, sqz mutants showed no other abnormality in transcription factor switching, whereas nab mutants showed the above-mentioned defects in transcription factor switching and timing of quiescence. These mutant phenotypes revealed that regulation of late temporal events is distributed into multiple pathways. Pdm probably coordinately regulates all factors involved in the timing of NB quiescence, because the loss of Pdm alone is sufficient to cause precocious entry into quiescence (Tsuji, 2008).

Nab and Sqz were shown to work for NB quiescence in NBs. The Nab/Sqz-mediated repression of Kr may be controlled in NBs due to changes in NB temporal identity, or in both NBs and their neurons. Nab might inhibit transcription by recruiting the nucleosome remodeling and deacetylase chromatin remodeling complex as does mammalian Nab (Srinivasan, 2006). Mammalian Nab acts with EGR-1, EGR-2 to determine the fate of cells in hematopoiesis (Laslo, 2006; Svaren, 1996), but whether it can act with the mammalian homolog of LIN-29/Sqz has not been reported. Loss of lin-29, a C. elegans homolog of sqz, causes a heterochronic phenotype in which adulthood is not reached and molting is repeated (Ambros, 1984; Rougvie, 1995). C. elegans has a nab homolog gene, mab-10, that acts with lin-29 in a heterochronic genetic cascade (Tsuji, 2008).

It is unclear what molecular mechanisms enable NBs to suspend the switching of transcription factor expression and maintain temporal identity during quiescence. It is known that the mechanisms for switching expression of early temporal transcription factors can be either cell division dependent or independent. Irrespective of the mechanism used, a NB can 'memorize' its temporal state before quiescence and resume the intrinsic temporal changes once cell cycle progression is reactivated. Embryonic stem cells may maintain multipotency during a slow proliferation state by staying in S phase. When quiescent NBs re-entered the cell cycle, their initial progeny incorporated BrdU fed since hatching, indicating that quiescent NBs stay either prior to S phase or early in S phase. It will be important to identify the point in the cell cycle at which NB enters quiescence (Tsuji, 2008).

Another well-established mechanism that governs temporal aspects of lineage formation is the heterochronic gene cascade in C. elegans. This cascade contains one each of the hunchback homolog and lin-29 genes and generates five distinct temporal cell identities within a single cell lineage. Drosophila NB lineage formation uses two types of Zn-finger proteins, namely the Hb/Cas class [Cas shares DNA-binding characteristics with Hb and the Kr/LIN-29 class. These pairs are expressed three times in NB lineages in the following order: (1) Hb and Kr-> (2) Cas, Kr and Sqz--> Cas and DmLin-29-->end of lineage. This sequence seems to produce at least ten distinct temporal identities within an NB lineage. The repetitive use of these temporal transcription factors in three distinct phases appears to have made the NB lineage longer and more diverse. Lack of Hb also generates NB lineage variety; the NB3-3 and NB2-1 lineages lack Hb and initiate their lineage with Kr. Although the model NB employed in this study lacks Hb, the sequence and entry into quiescence described in this study are common to many typical NB lineages that start with Hb. Interesting questions from the perspective of evolution are how do the three phases combine to form a single lineage and how has NB quiescence evolved in the middle of the NB lineages (Tsuji, 2008)?

Neural stem cells in the mouse cerebral cortex go through ~11 divisions and some enter quiescence in late embryo. The possibility has to be considered that mammalian neural stem cell and Drosophila NB share a similar intrinsic mechanism that induces quiescence (Tsuji, 2008).

Developmental transcriptional networks are required to maintain neuronal subtype identity in the mature nervous system

During neurogenesis, transcription factors combinatorially specify neuronal fates and then differentiate subtype identities by inducing subtype-specific gene expression profiles. But how is neuronal subtype identity maintained in mature neurons? Modeling this question in two Drosophila neuronal subtypes (Tv1 and Tv4), tests were performed to see whether the subtype transcription factor networks that direct differentiation during development are required persistently for long-term maintenance of subtype identity. By conditional transcription factor knockdown in adult Tv neurons after normal development, it was found that most transcription factors within the Tv1/Tv4 subtype transcription networks are indeed required to maintain Tv1/Tv4 subtype-specific gene expression in adults. Thus, gene expression profiles are not simply 'locked-in,' but must be actively maintained by persistent developmental transcription factor networks. The cross-regulatory relationships were examined between all transcription factors that persisted in adult Tv1/Tv4 neurons. Certain critical cross-regulatory relationships that had existed between these transcription factors during development are no longer present in the mature adult neuron. This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons. Together, these results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors. These findings have implications for understanding the maintenance of all long-lived cell types and the functional degeneration of neurons in the aging brain (Eade, 2012).

The data provide novel insight supporting the view of Blau and Baltimore (1991) that cellular differentiation is a persistent process that requires active maintenance, rather than being passively 'locked-in' or unalterable. Two primary findings are made in this study regarding the long-term maintenance of neuronal identity. First, all known developmental transcription factors acting in postmitotic Tv1 and Tv4 neurons to initiate the expression of subtype terminal differentiation genes are then persistently required to maintain their expression. Second, it was found that key developmental cross-regulatory relationships that initiated the expression of certain transcription factors were no longer required for their maintained expression in adults. Notably, this was found to be the case even between transcription factors whose expression persists in adults (Eade, 2012).

In this study, all transcription factors implicated in the initiation of subtype-specific neuropeptide expression in Tv1 and Tv4 neurons were found to maintain subtype terminal differentiation gene expression in adults (see Summary of changes in subtype transcription network configuration between initiation and maintenance of subtype identity). In Tv1, col, eya, ap and dimm are required for Nplp1 initiation during development. In this study, knockdown of each transcription factor in adult Tv1 neurons was shown to dramatically downregulate Nplp1. In Tv4 neurons, FMRFa initiation during development requires eya, ap, sqz, dac, dimm and retrograde BMP signaling. Together with previous work showing that BMP signaling maintains FMRFa expression in adults (Eade, 2009), this study now demonstrates that all six regulatory inputs are required for FMRFa maintenance. Most transcription factors, except for dac, also retained their relative regulatory input for FMRFa and Nplp1 expression. In addition, individual transcription factors also retained their developmental subroutines. For example, as found during development, dimm was required in adults to maintain PHM (independently of other regulators) and FMRFa/Nplp1 expression (combinatorially with other regulators) (Eade, 2012).

The few genetic studies that test a persistent role for developmental transcription factors support their role in initiating and maintaining terminal differentiation gene expression. In C. elegans, where just one or two transcription factors initiate most neuronal subtype-specific terminal differentiation genes, they then also appear to maintain their target terminal differentiation genes. In ASE and dopaminergic neurons respectively, CHE-1 and AST-1 initiate and maintain expression of pertinent subtype-specific terminal differentiation genes. In vertebrate neurons, where there is increased complexity in the combinatorial activity of transcription factors in subtype-specific gene expression, certain transcription factors have been demonstrated to be required for maintenance of subtype identity. These are Hand2 that initiates and maintains tyrosine hydroxylase and dopa fl-hydroxylase expression in mouse sympathetic neurons, Pet-1, Gata3 and Lmx1b for serotonergic marker expression in mouse serotonergic neurons, and Nurr1 for dopaminergic marker expression in murine dopaminergic neurons (Eade, 2012).

However, while these studies confirm a role for certain developmental transcription factors in subtype maintenance, it had remained unclear whether the elaborate developmental subtype transcription networks, that mediate neuronal differentiation in Drosophila and vertebrates, are retained in their entirety for maintenance, or whether they become greatly simplified. This analysis of all known subtype transcription network factors in Tv1 and Tv4 neurons now indicates that the majority of a developmental subtype transcription network is indeed retained and required for maintenance. Why would an entire network of transcription factors be required to maintain subtype-specific gene expression? The combinatorial nature of subtype-specific gene expression entails cooperative transcription factor binding at clustered cognate DNA sequences and/or synergism in their activation of transcription. In such cases, the data would indicate that this is not dispensed with for maintaining terminal differentiation gene expression in mature neurons (Eade, 2012).

How the transcription factors of the subtype transcription networks are maintained is less well understood. An elegant model has emerged from studies in C. elegans, wherein transcription factors stably auto-maintain their own expression and can then maintain the expression of subtype terminal differentiation genes. The transcription factor CHE-1 is a key transcription factor that initiates and maintains subtype identity in ASE neurons. CHE-1 binds to a cognate DNA sequence motif (the ASE motif) in most terminal differentiation genes expressed in ASE neurons, as well as in its own cis-regulatory region. Notably, a promoter fusion of the che-1 transcription factor failed to express in che-1 mutants, indicative of CHE-1 autoregulation, and for the cooperatively-acting TTX-3 and CEH-10 transcription factors in AIY neurons. Thus, subtype maintenance in C. elegans is anchored by auto-maintenance of the transcription factors that initiate and maintain terminal differentiation gene expression (Eade, 2012).

In contrast, all available evidence in Tv1 and Tv4 neurons fails to support such an autoregulatory mechanism. An ap reporter (apC-t-lacZ) is expressed normally in ap mutants, and in this study apdsRNAi was not found to alter apGAL4 reporter activity. Moreover, col transcription was unaffected in col mutants that express a non-functional Col protein. This leaves unresolved the question of how the majority of the transcription factors are stably maintained. For transcription factors that are initiated by transiently expressed inputs, a shift to distinct maintenance mechanisms have been invoked and in certain cases shown. In this study, this was found for the loss of cas expression in Tv1 (required for col initiation) and the loss of cas, col and grh in Tv4 (required for eya, ap, dimm, sqz, dac initiation). However, it was surprising to find that the cross-regulatory relationships between persistently-expressed transcription factors were also significantly altered in adults. Notably, eya initiated but did not maintain dimm in Tv4. In Tv1, col initiated but did not maintain eya, ap or dimm. This was particularly unexpected as eya remained critical for FMRFa maintenance and col remained critical for Nplp1 maintenance. Indeed, although tests were performed for cross-regulatory interactions between all transcription factors in both the Tv1 and Tv4 subtype transcription networks, only Dimm was found to remain dependent upon its developmental input; Eya and Ap in Tv1 as well as Ap in Tv4. However, even in this case, the regulation of Dimm was changed; it no longer required eya in Tv4, and in Tv1 it no longer required col, in spite of the fact that both col and eya are retained in these neurons. It is anticipated that such changes in transcription factor cross-regulatory relationships will be found in other Drosophila and vertebrate neurons, which exhibit high complexity in their subtype transcription networks. Indeed, recent evidence has found that in murine serotonergic neurons, the initiation of Pet-1 requires Lmx-1b, but ablation of Lmx-1b in adults did not perturb the maintenance of Pet-1 expression (Eade, 2012).

The potential role of autoregulation for the other factors in the Tv1/Tv4 subtype transcription networks is being pursued. However, there are three additional, potentially overlapping, models for subtype transcription network maintenance. First, regulators may act increasingly redundantly upon one another. Second, unknown regulators may become increasingly sufficient for transcription factor maintenance. Third, transcription factors may be maintained by dedicated maintenance mechanisms, as has been shown for the role of trithorax group genes in the maintenance of Hox genes and Engrailed. Moreover, chromatin modification is undoubtedly involved and likely required to maintain high-level transcription of Tv transcription factors as well as FMRFa, Nplp1 and PHM. However, the extent to which these are instructive as opposed to permissive has yet to be established. In this light, it is intriguing that MYST-HAT complexes, in addition to the subtype transcription factors Che-1 and Die-1, are required for maintenance of ASE-Left subtype identity in C. elegans (Eade, 2012).

Taken together, these studies have identified two apparent types of maintenance mechanism that are operational in adult neurons. On one hand, there are sets of genes that are maintained by their initiating set of transcription factors. These include the terminal differentiation genes and the transcription factor dimm. On the other, most transcription factors appear to no longer require regulatory input from their initiating transcription factor(s). Further work will be required to better understand whether these differences represent truly distinct modes of gene maintenance or reflect the existence of yet unidentified regulatory inputs onto these transcription factors. One issue to consider here is that the expression of certain terminal differentiation genes in neurons, but perhaps not subtype transcription factors, can be plastic throughout life, with changes commonly occurring in response to a developmental switch or physiological stimulus. Thus, terminal differentiation genes may retain complex transcriptional control in order to remain responsive to change. It is notable, however, that FMRFa, Nplp1 and PHM appear to be stably expressed at high levels in Tv1/4 neurons, and no conditions were found that alter their expression throughout life. Thus, these are considered to be stable terminal differentiation genes akin to serotonergic or dopaminergic markers in their respective neurons that define those cells' functional identity and, where tested, are actively maintained by their developmental inputs. Tv1/4 neurons undoubtedly express a battery of terminal differentiation genes, and sets of unknown transcription factors are likely required for their subtype-specific expression. Subtype transcription networks are considered to encompass all regulators required for differentiating the expression of all subtype-specific terminal differentiation genes. Further, differentiation of subtype identity is viewed as the completion of a multitude of distinct gene regulatory events in which each gene is regulated by a subset of the overall subtype transcription network. As highly restricted terminal differentiation genes expressed in Tv1 and Tv4 neurons, it is believed that Nplp1, FMRFa and PHM provide a suitable model for the maintenance of overall identity, with the understanding that other unknown terminal differentiation genes expressed in Tv1 and Tv4 may not be perturbed by knockdown of the transcription factors tested in this study. In the future, it will be important to incorporate a more comprehensive list of regulators and terminal differentiation genes for each neuronal subtype. However, it is believed that the principles uncovered in this study for FMRFa, Nplp1 and PHM maintenance will hold for other terminal differentiation genes (Eade, 2012).

Finally, it is proposed that the active mechanisms utilized for maintenance of subtype differentiation represent an Achilles heel that renders long-lived neurons susceptible to degenerative disorders. Nurr1 ablation in adult mDA neurons reduced dopaminergic markers and promoted cell death. Notably, Nurr1 mutation is associated with Parkinson's disease, and its downregulation is observed in Parkinson's disease mDA neurons. Adult mDA are also susceptible to degeneration in foxa2 heterozygotes, another regulator of mDA neuron differentiation that is maintained in adult mDA neurons. Studies in other long-lived cell types draw similar conclusions. Adult conditional knockout of Pdx1 reduced insulin and fl-cell mass and, importantly, heterozygosity for Pdx1 leads to a rare monogenic form of non-immune diabetes, MODY4. Similarly, NeuroD1 haploinsufficiency is linked to MODY6 and adult ablation of NeuroD in β-islet cells results in β-cell dysfunction and diabetes. These data, together with current results, underscore the need to further explore the transcriptional networks that actively maintain subtype identity, and hence the function, of adult and aging cells (Eade, 2012).


EVOLUTIONARY HOMOLOGS

MAB-10/NAB acts with LIN-29/EGR to regulate terminal differentiation and the transition from larva to adult in C. elegans

In Caenorhabditis elegans, a well-defined pathway of heterochronic genes ensures the proper timing of stage-specific developmental events. During the final larval stage, an upregulation of the let-7 microRNA indirectly activates the terminal differentiation factor and central regulator of the larval-to-adult transition, LIN-29, via the downregulation of the let-7 target genes lin-41 and hbl-1. This study identifies a new heterochronic gene, mab-10, and shows that mab-10 encodes a NAB (NGFI-A-binding protein) transcriptional co-factor. MAB-10 acts with LIN-29 to control the expression of genes required to regulate a subset of differentiation events during the larval-to-adult transition, and the NAB-interaction domain of LIN-29 is conserved in Kruppel-family EGR (early growth response) proteins. A similar interaction between Drosophila NAB and the two Drosophila LIN-29 homologs RN and SQZ was reported recently. In mammals, EGR proteins control the differentiation of multiple cell lineages, and EGR-1 acts with NAB proteins to initiate menarche by regulating the transcription of the luteinizing hormone β subunit. Genome-wide association studies of humans and various studies of mouse recently have implicated the mammalian homologs of the C. elegans heterochronic gene lin-28 in regulating cellular differentiation and the timing of menarche. This work suggests that human homologs of multiple C. elegans heterochronic genes might act in an evolutionarily conserved pathway to promote cellular differentiation and the onset of puberty (Harris, 2011).

This study identified mab-10 as a new heterochronic gene that is required for specific aspects of the larval-to-adult transition, specifically molting cycle exit and seam cell exit from the cell cycle. mab-10 encodes the only C. elegans NAB transcriptional co-factor. NAB proteins are thought to physically interact with Kruppel family EGR transcription factors to regulate their activity (Harris, 2011).

Previous work demonstrated that MAB-10 (then known only as the C. elegans NAB protein R166.1) could interact with mammalian EGR proteins in a yeast two-hybrid assay; no corresponding C. elegans EGR protein was identified. This study has demonstrate that MAB-10 interacts with the terminal differentiation factor LIN-29 through an evolutionarily conserved NAB binding domain (R1 domain) and that MAB-10 is required for a subset of LIN-29-dependent activities. This work identifies LIN-29 as a C. elegans EGR-like protein and demonstrates that the C. elegans heterochronic pathway controls the timing of NAB/EGR-mediated differentiation (Harris, 2011).

Several experiments using mammalian tissue culture suggest that NAB proteins negatively regulate EGR activity by binding EGR proteins at specific target genes and preventing EGR-mediated transcription. However, loss of either EGR2 function or NAB function in mice and humans results in hypomyelination, suggesting that EGR and NAB proteins need not act antagonistically in vivo (Harris, 2011).

In C. elegans, MAB-10 and LIN-29 both act to promote terminal differentiation and the onset of adulthood. Furthermore, mab-10 promotes the formation of precocious adult alae in a lin-41 mutant background, suggesting that MAB-10 does not specifically act to control genes required for exit from the molting cycle and seam cell exit from the cell cycle, but more likely acts as a general enhancer of LIN-29 activity (Harris, 2011).

EGR and NAB proteins have been shown to operate in a negative-feedback loop wherein an EGR protein promotes the expression of its NAB co-factor, which then inhibits EGR activity. mab-10 transcription does not depend on LIN-29, despite a dramatic increase of mab-10 transcription during the L4 stage. Thus, mab-10 is not a transcriptional target of LIN-29 (Harris, 2011).

Whereas mab-10 is not a transcriptional target of LIN-29, MAB-10::GFP localization to seam cell nuclei during the L4 stage required LIN-29, indicating that LIN-29 might promote MAB-10 seam cell nuclear localization via a post-transcriptional mechanism or via direct physical interaction (Harris, 2011).

This work demonstrates that MAB-10 and LIN-29 do not operate in a negative-feedback loop. It is proposed that other components of the heterochronic pathway directly regulate mab-10 transcription to temporally regulate MAB-10/LIN-29 activity and that LIN-29 or some factor downstream of LIN-29 controls MAB-10/LIN-29 activity by promoting the accumulation of MAB-10 in seam cell nuclei (Harris, 2011).

By showing that MAB-10 acts with LIN-29 through an evolutionarily conserved EGR R1 domain, LIN-29 and the Drosophila LIN-29 homologs RN and SQZ are identified as EGR-like molecules. It is proposed that NAB proteins and EGR proteins act together in temporal developmental programs to control terminal differentiation. In Drosophila, the LIN-29 homolog SQZ acts with Drosophila NAB to control neuroblast differentiation. In C. elegans, LIN-29 and MAB-10 act together to control the differentiation of a hypodermal stem cell lineage during the transition from larva to adult by regulating the expression of the nuclear hormone receptors nhr-23 and nhr-25 and the cell cycle regulator cki-1. Recently, a study of C. elegans demonstrated that nhr-25 is itself a heterochronic gene and possibly functions with lin-29 to promote aspects of the larval-to-adult transition, including seam cell exit from the cell cycle. Though the mechanism by which nhr-25 regulates seam cell exit from the cell cycle is not known, it is speculated that LIN-29 and NHR-25 might act together to promote cki-1 expression (Harris, 2011).

EGR proteins were originally identified as immediate-early genes and generally have been regarded as differentiation factors. Like mab-10 and lin-29 mutants, Nab and Egr mutant mice are defective in the terminal differentiation of several cell lineages. For example, in Schwann cells, EGR2 promotes the expression of P27, the homolog of C. elegans CKI-1, and acts with NAB proteins to promote terminal differentiation. Mammalian homologs of other C. elegans heterochronic genes also control differentiation. Similar to the role of LIN-28 in C. elegans, mammalian LIN28 and LIN28B promote stem cell identity and prevent differentiation by repressing the let-7 microRNA gene. As in C. elegans, increasing levels of let-7 drive differentiation, and the mouse homolog of LIN-41, LIN41, has been shown to be a let-7 target acting in stem cell niches to prevent premature differentiation (Harris, 2011).

Mammalian LIN-28 controls the timing of the onset of puberty in mice and possibly humans. Mice lacking EGR1 function, like lin-29 mutants of C. elegans, fail to undergo puberty. EGR1 and NAB proteins act with SF1, the homolog of C. elegans NHR-25, in the gonadotrope lineage of the pituitary gland to regulate the expression of luteinizing hormone and the onset of puberty. The molecular mechanism by which mammalian LIN-28 regulates the onset of puberty is not known. This work raises the possibility that homologs of C. elegans heterochronic genes might act in an evolutionarily conserved pathway that controls the terminal differentiation of cell lineages and the onset of adulthood by regulating the activity of NAB and EGR proteins (Harris, 2011).


REFERENCES

Search PubMed for articles about Drosophila squeeze

Allan, D. W., St Pierre, S. E., Miguel-Aliaga, I. and Thor, S. (2003). Specification of neuropeptide cell identity by the integration of retrograde BMP signaling and a combinatorial transcription factor code. Cell 113(1): 73-86. Medline abstract: 12679036

Allan, D. W., Park, D., St. Pierre, S. E., Taghert, P. H. and Thor, S. (2005). Regulators acting in combinatorial codes also act independently in single differentiating neurons. Neuron 45: 689-700. Medline abstract: 15748845

Ambros, V. and Horvitz, H. R. (1984). Heterochronic mutants of the nematode Caenorhabditis elegans. Science 226: 409-416. PubMed Citation: 6494891

Berman, B. P., et al. (2004). Computational identification of developmental enhancers: conservation and function of transcription factor binding-site clusters in Drosophila melanogaster and Drosophila pseudoobscura. Genome Biol. 5(9): R61. Medline abstract: 15345045

Blau, H. M. and Baltimore, D. (1991). Differentiation requires continuous regulation. J. Cell Biol. 112: 781-783. PubMed Citation: 1999456

Clements, M., Duncan, D. and Milbrandt, J. (2003). Drosophila NAB (dNAB) is an orphan transcriptional co-repressor required for correct CNS and eye development. Dev. Dyn. 226: 67-81. Medline abstract: 12508226

Eade, K. T. and Allan, D. W. (2009). Neuronal phenotype in the mature nervous system is maintained by persistent retrograde bone morphogenetic protein signaling. J. Neurosci. 29: 3852-3864. PubMed Citation: 19321782

Eade, K. T., Fancher, H. A., Ridyard, M. S. and Allan, D. W. (2012). Developmental transcriptional networks are required to maintain neuronal subtype identity in the mature nervous system. PLoS Genet. 8(2): e1002501. PubMed Citation: 22383890

Herrero, P., et al. (2007). Squeeze involvement in the specification of Drosophila leucokinergic neurons: Different regulatory mechanisms endow the same neuropeptide selection. Mech. Dev. 124: 427-440. Medline abstract: 17442544

Felix, J. T., Magarinos, M. and Diaz-Benjumea, F. J. (2007). Nab controls the activity of the zinc-finger transcription factors Squeeze and Rotund in Drosophila development. Development 134(10): 1845-52. Medline abstract: 17428824

Harris, D. T. and Horvitz, H. R. (2011). MAB-10/NAB acts with LIN-29/EGR to regulate terminal differentiation and the transition from larva to adult in C. elegans. Development 138(18): 4051-62. PubMed Citation: 21862562

Komiyama, T. and Luo, L. (2007). Intrinsic control of precise dendritic targeting by an ensemble of transcription factors. Curr. Biol. 17(3): 278-85. Medline abstract: 17276922

Laslo, P., Spooner, C. J., Warmflash, A., Lancki, D. W., Lee, H. J., Sciammas, R., Gantner, B. N., Dinner, A. R. and Singh, H. (2006). Multilineage transcriptional priming and determination of alternate hematopoietic cell fates. Cell 126: 755-766. PubMed Citation: 16923394

Le, N., Nagarajan, R., Wang, J. Y., Svaren, J., LaPash, C., Araki, T., Schmidt, R. E. and Milbrandt, J. (2005). Nab proteins are essential for peripheral nervous system myelination. Nat. Neurosci. 8: 932-940. Medline abstract: 16136673

Mechta-Grigoriou, F., Garel, S. and Charnay, P. (2000). Nab proteins mediate a negative feedback loop controlling Krox-20 activity in the developing hindbrain. Development 127: 119-128. Medline abstract: 10654606

Rougvie, A. E. and Ambros, V. (1995). The heterochronic gene lin-29 encodes a zinc finger protein that controls a terminal differentiation event in Caenorhabditis elegans. Development 121: 2491-2500. PubMed Citation: 7671813

Sevetson, B. R., Svaren, J. and Milbrandt, J. (2000). A novel activation function for NAB proteins in EGR-dependent transcription of the luteinizing hormone beta gene. J. Biol. Chem. 275: 9749-9757. Medline abstract: 10734128

Srinivasan, R., Mager, G. M., Ward, R. M., Mayer, J. and Svaren, J. (2006). NAB2 represses transcription by interacting with the CHD4 subunit of the nucleosome remodeling and deacetylase (NuRD) complex. J. Biol. Chem. 281: 15129-15137. PubMed Citation: 16574654

Svaren, J., Sevetson, B. R., Apel, E. D., Zimonjic, D. B., Popescu, N. C. and Milbrandt, J. (1996). NAB2, a corepressor of NGFI-A (Egr-1) and Krox20, is induced by proliferative and differentiative stimuli. Mol. Cell. Biol. 16: 3545-3553. PubMed Citation: 8668170

Svaren, J., Sevetson, B. R., Golda, T., Stanton, J. J., Swirnoff, A. H. and Milbrandt, J. (1998). Novel mutants of NAB corepressors enhance activation by Egr transactivators. EMBO J. 17: 6010-6019. Medline abstract: 9774344

Swirnoff, A. H., Apel, E. D., Svaren, J., Sevetson, B. R., Zimonjic, D. B., Popescu, N. C. and Milbrandt, J. (1998). Nab1, a corepressor of NGFI-A (Egr-1), contains an active transcriptional repression domain. Mol. Cell. Biol. 18: 512-524. Medline abstract: 9418898

Tsuji, T., Hasegawa, E. and Isshiki, T. (2008). Neuroblast entry into quiescence is regulated intrinsically by the combined action of spatial Hox proteins and temporal identity factors. Development 135(23): 3859-69. PubMed Citation: 18948419


Biological Overview

date revised: 15 December 2011

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